Revealing the Regulatory Network of Stem Secondary Growth in Populus

In plants, Class I KNOX and Class III HD ZIP transcription factors are critical regulators of stem secondary growth and wood formation, which is originated from the lateral meristem - vascular cambium. However, very little is known at the molecular level about how these transcription factors participate in the regulation of secondary growth. Here, we describe the use of next generation sequencing-based genomic applications to develop genome-wide datasets that can be used to investigate direct and non-direct targets of Class I KNOX and class III HD ZIP transcription factors. In this talk, we present a case study for the Class I KNOX ARBORKNOX1 (ARK1). First, we developed a ChiP-seq (Chromatin immunoprecipitation coupled with high-throughput sequencing) protocol for stem tissues of Populus. In our strategy, we designed two unique antibodies that recognize peptides specific to each transcription factor of interest, and use those antibodies for ChIP. DNA fragments from ChIP experiments were then used to prepare sequencing libraries and finally mapped to Populus genome. The use of multiple antibodies against the same transcription factor allows cross-validation of antibody performance and putative binding loci across the whole genome. Comparing replicates of ARK1 ChIP-seq results between two different antibodies give around 70% overlapping peaks of the binding loci, indicating these ChIP-seq data are reliable. Next, we used RNA-seq to study the differentially expressed genes between wild type vs. ARK1 overexpression mutants. An analysis with ARK1 ChIP-binding loci and differentially expressed genes from RNA-seq will be presented.